Project Summary High altitude residence (HA, >2500 m) increases the frequency of preeclampsia (PreE) and intrauterine growth restriction (IUGR) 3-fold. Since hypoxia is a common cause, HA studies are uniquely positioned to evaluate the mechanistic role of hypoxia, discover new treatments, and alleviate the ?almost complete lack of drug development for obstetric indications? noted in PAR-13-389. Adenosine monophosphate kinase (AMPK) is a regulator of metabolic homeostasis that also affects vascular growth and function under hypoxia. We have shown that AMPK plays important roles in the regulation of human uterine artery (UtA) blood flow and fetal growth at HA, and that AMPK activation has vasodilator effects in isolated murine UtA that are potentiated by hypoxia. Our central hypothesis is that AMPK activation promotes vasodilation in the uteroplacental circulation in response to hypoxia to raise UtA blood flow and improve fetal growth at HA. Because hypoxia leads to epigenetic changes that alter the expression of genes regulating of AMPK activity and fetal growth, we propose that DNA methylation influences these AMPK-mediated processes. We present new murine and human data to show that hypoxia a) raises placental phosphorylated (P)- relative to total AMPK levels and the P- as well as the total protein levels of AMPK targets in thoracic aorta and placenta; b) increases the expression of key enzymes activating AMPK in UtA and placenta; and c) alters methylation-expression relationships of AMPK- signaling genes important for fetal growth. We will test our central hypothesis by determining: 1. In Aim 1, the effect of HA pregnancy on AMPK signaling and its relationship to UtA blood flow and fetal growth in humans. We will recruit 102 healthy residents of low altitude (LA, 1600 m, n=53) or HA (3000 m, n=49); measure UtA blood flow and fetal biometry longitudinally; and determine the activation of AMPK, its upstream regulators and downstream targets, the expression levels and DNA methylation of relevant genes in peripheral blood mononuclear cells (PBMCs), and plasma levels of AMPK regulators. 2. In Aim 2, the effects of hypoxia on AMPK activation in human myometrial artery (MA) and placenta, and on MA vasoreactivity. In women participating in Aim 1 who deliver by elective C-section (n=19/altitude), we will obtain myometrial biopsies and placentas to determine a) the activation, expression and DNA methylation status of AMPK, its well-established regulators and downstream targets; b) the effect of HA pregnancy on MA vasoreactivity; and c) whether hypoxia potentiates vasodilator effects of AMPK activation in MA. 3. In Aim 3, the role of AMPK in regulating uteroplacental blood flow and fetal growth in response to hypoxia in mice. Pregnant mice housed at sea level (SL) or HA will be treated with the AMPK activator (AICAR), inhibitor (Compound C), or vehicle (control) for determining the separate and combined effects of hypoxia and AMPK activation on AMPK signaling in UtA, placental and fetal tissues; UtA vascular reactivity and blood flow; and fetal growth.